The determination of enzyme activities (or substrate concentrations), especially in blood serum or plasma, plays an important role in clinical chemical diagnostics. Test procedures are often used for this which are based on the reduction of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) and photometric detection of the resulting change of the absorption behaviour in the ultraviolet wavelength range (λ=334, 340 or 365 nm). When suitable test conditions have been selected, this change is linearly proportional to the enzyme activity (or substrate concentration) to be determined.
Nowadays the methods described in Eur. J. Clin. Chem. Clin. Biochem. 31, 897 (1994) and Eur. J. Clin. Chem. Clin. Biochem. 32, 639 (1994) are generally recommended for determining the enzyme activity of for example lactate dehydrogenase (LDH, E.C.1.1.1.27). The test principle involves the oxidation of lactate to pyruvate while a coenzyme such as NAD or NADP is simultaneously reduced to NADH or NADPH. Such a conversion, in this case is for example catalysed by LDH, takes place in an alkaline medium (pH 9.4). As a result of this instability there is a relatively rapid increase in absorbance (the so-called reagent blank) in the wavelength range for the measurement and hence the reagent combination becomes unusable already after a short time (3 months) even when stored in a refrigerator (2° to 8° C.). This is a particular problem for the production of ready-to-use liquid reagents with a long shelf-life which are intended to enable the user to carry out analyses in the daily routine in a simple and reliable manner.
A method for stabilizing aqueous coenzymes using chelating agents and azides is known from JP 84/82398. However, a disadvantage of this method is that it is necessary to add azide which is nowadays classified as cancerogenic and which, moreover, has an inhibitory effect on many enzymes.
It is also known that coenzyme solutions can be stabilized by adding heavy metal salts, for example in the form of copper (II) ions, and thus prevent an increase of the reagent blank (DE 195 43 493 or EP 0 804 610). However, degradation products may form during long storage periods or at high storage temperatures (already above 10° C.) which inhibit the dehydrogenase enzyme to be determined and thus result in measured values that are too low. A reagent that can be stabilized over a long period (3 months and more) of constant quality which thus, among other things, does not require repeated calibrations is not available at present.